Andrew P. Supp

Assessment of a silver-coated barrier dressing for potential use with skin grafts on excised burns

Acticoat burn dressing is a silver-coated dressing with antimicrobial activity purported to reduce infection from environmental organisms in partial and full-thickness wounds. Acticoat was tested for activity as an antimicrobial treatment and as an antimicrobial barrier dressing in three in vitro assays. It was found that a modified disc assay method gave false negative results but in an assay in which bacteria were inoculated on top of samples of Acticoat, bacterial numbers were reduced, over time, with all microorganisms tested. Acticoat served as a barrier for bacteria, inoculated onto it, from contaminating the surface of an agar plate under the Acticoat. The data show that Acticoat has: antimicrobial capabilities, but to be effective hours of contact between Acticoat and the microorganisms are required; and the capacity to serve as an antimicrobial barrier dressing. These findings support the conclusion that Acticoat has activity to reduce microbial contamination of wounds from environmental sources.

Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patients self‐image. To address these morbidities, human fibroblasts were inoculated on a collagen‐glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 × 106/cm2 combined with either 0, 1.1 × 102, 1.1 × 103, or 1.1 × 104 melanocytes/cm2. The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L‐Dopa–positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL‐5. The skin substitutes with 1.1 × 104 melanocytes/cm2 were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 × 103 melanocytes/cm2 group, to complete pigmentation in the 1.1 × 104 melanocytes/cm2 group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 × 103 and 1.1 × 104 melanocytes/cm2. These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 × 104 melanocytes/cm2 to skin substitutes. (WOUND REP REG 2002;10:378–386)

Glutaraldehyde crosslinking of collagen substrates inhibits degradation in skin substitutes grafted to athymic mice

Collagen-based implants have been described as vehicles for transplantation of cultured skin cells for treatment of burn wounds. To optimize vascularization and repair of connective tissue, collagen solubility and glutaraldehyde crosslinking were evaluated. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates that were prepared from acid-insoluble, or partially soluble collagen. Substrates were crosslinked with 0% or 0.25% glutaraldehyde, populated with cells, and grafted to full-thickness wounds on athymic mice (n = 6/condition). After 6 weeks, the wound area was measured by planimetry, and healed wounds were scored by histochemistry for immunoreactivity to HLA-ABC and bovine collagen. Data analysis shows that crosslinking of collagen implants with glutaraldehyde is associated (p < 0.001) with detection of the implant. No association was found between solubility of bovine collagen and immunodetection. Epidermis of all wounds was positive for HLA-ABC, and no differences in wound areas were found. These results suggest that glutaraldehyde crosslinking of collagen implants decreases the rate of biodegradation. Delayed degradation of crosslinked collagen may result clinically in reduced engraftment of skin substitutes.

Assessment with the dermal torque meter of skin pliability after treatment of burns with cultured skin substitutes.

The assessment of visco-elastic (V-E) properties in cutaneous scars is critical to reduction of impairment and restoration of function after grafting of excised burns. Cultured skin substitutes (CSS) that consist of autologous keratinocytes and fibroblasts attached to biopolymer substrates are alternatives for permanent closure of excised, full-thickness burns, but assessment of scarring has been subjective. V-E properties of CSS were measured with a Dia-Stron Dermal Torque Meter (DTM 310, Dia-Stron, Ltd, Broomall, Pa), which applies a constant torque (10 mNm) for a fixed interval (10 seconds) and measures rotational deformation and recovery. Parameters of skin deformation were measured in patients (n = 10) after grafting of CSS or meshed skin autograft. Native human skin (NHS) of healthy volunteers (n = 13) served as the control. Skin healed after treatment with CSS or autograft was evaluated for 1 year or longer after grafting. Elastic stretch (Ue), viscous stretch (Uv), total extensibility (Uf), elastic recovery (Ur), total recovery (Ua), and residual plasticity (R) were measured as degrees of rotation, were tested for significance (P < .05) by Student t test comparisons between treatment groups and controls, and were subjected to regression analysis. Assessment of burn scar with the Dermal Torque Meter detected time-dependent increases of all individual parameters of V-E properties for both CSS and autograft during the first year after grafting. At 1 year or later, no statistical differences were found between CSS and autograft for individual parameters, but Ue and Ur for autograft were significantly lower than for NHS. At 1 year or longer, autograft was significantly different from CSS or NHS, with a greater ratio of Uv to Ue, and both graft types had a lower ratio of Ur to Uf than NHS had. These results suggest that instrumental measurement of scar pliability may increase objectivity in assessment of patient recovery and establish an absolute scale for quantitative analysis of V-E properties in skin after grafting of conventional or alternative skin substitutes.

Surface electrical capacitance as an index of epidermal barrier properties of composite skin substitutes and skin autografts

Restoration of the epidermal barrier is a requirement for burn wound closure. A rapid, reliable, and noninvasive measure of the rate of restoration of the epidermal barrier is not readily available. To monitor the reformation of the epidermal barrier, we measured surface electrical capacitance on cultured skin substitutes (human keratinocytes and fibroblasts attached to collagen‐glycosaminoglycan substrates) and split‐thickness skin autografts grafted to patients. Data were collected from four patients with burns and one pediatric patient with a congenital hairy nevus comprising > 60% total body surface area. Capacitance measurements were performed at days 7, 10, 12, 14, and 28 by direct contact of the capacitance probe for 10 seconds to the cultured skin substitutes or split‐thickness autograft. On postoperative days 7, 10, 12, 14, 21, and 28, the surface electrical capacitance of cultured skin substitutes after 10 seconds of sampling was 2468 ± 268, 1443 ± 439, 129 ± 43, 200 ± 44, 88 ± 20, and 74 ± 19 picofarads (mean ± standard error of the mean), respectively. Surface electrical capacitance for split‐thickness autograft on the same days was 1699 ± 371, 1914 ± 433, 125 ± 16, 175 ± 63, 110 ± 26, 271 ± 77 picofarads, respectively. Surface electrical capacitance in all of the grafts decreased with time. Cultured skin substitutes had approximately the same 10‐second capacitance values as split‐thickness autograft during 3 weeks of healing and approached values for uninjured skin (32 ± 5 picofarads) by 12 days. Measurement of surface electrical capacitance is a direct, inexpensive, and convenient index for noninvasive monitoring of epidermal barrier formation.

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice.

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen‐glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air–liquid interface in ambient (48–61%) or saturated (79–91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA™ Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.

Expression of interleukin-1alpha, interleukin-6, and basic fibroblast growth factor by cultured skin substitutes before and after grafting to full-thickness wounds in athymic mice.

OBJECTIVES Cultured skin substitutes (CSSs), consisting of human keratinocytes and human fibroblasts attached to collagen-glycosaminoglycan substrates, have been demonstrated to cover wounds, and may release detectable quantities of growth factors that promote wound healing. MATERIALS AND METHODS Basic fibroblast growth factor (bFGF), interleukin-1alpha (IL-1alpha), and interleukin-6 (IL-6) were assayed by enzyme linked immunosorbent assay and immunohistochemistry in CSSs in vitro and at days 1, 3, 7, 14, and 21 after grafting to full-thickness wounds in athymic mice. MEASUREMENTS AND MAIN RESULTS When isolated cells were tested, IL-1alpha was found to come primarily from the keratinocytes, whereas bFGF was from the fibroblasts. Combinations of both cell types in the CSSs resulted in a synergistic enhancement of IL-6 expression. Quantities of all three cytokines from CSSs were greater in vitro compared with in vivo levels at all time points after grafting. bFGF increased from day 1 to day 7, and then remained relatively constant until day 21. At day 3 maximal levels of IL-1alpha were observed. By day 7, IL-1alpha decreased to approximately 40% of maximal levels, and subsequently increased until day 21. IL-6 levels were highest at day 7 after grafting. All cytokines had reached elevated levels during the time of wound revascularization (days 3-7). CONCLUSIONS The sequence of cytokine synthesis in the wounds (i.e., rapid IL-1alpha increase followed by IL-6 expression) parallels serum levels reported after a septic challenge. These findings support the hypothesis that the wound is a source of systemic cytokines.

Delivery and activity of antimicrobial drugs released from human fibrin sealant.

Engraftment and healing of native or cultured skin grafts depend on adherence, vascularization, and control of microbial contamination in the wound bed. Fibrin sealant is a biocompatible polymer that may be used to promote skin engraftment by serving as a delivery vehicle for antimicrobial drugs. Human fibrin sealant (25 mg/ml) was polymerized with antibacterial agents (mupirocin [32 micrograms/ml], nitrofurazone [0.02% wt/vol], polymyxin B [400 U/ml], or norfloxacin [20 micrograms/ml]) on nitrocellulose (nc) backing and was prepared as 6 mm diameter discs with skin punches. Discs (n = 6) were applied in the Wet Disc Assay to clinical isolates of Staphylococcus aureus (mupirocin, nitrofurazone) or Pseudomonas aeruginosa (polymyxin B, norfloxacin). Controls included drug applied to 6 mm paper discs (25 microliter) and nitrocellulose discs submerged in each drug, blotted, and applied to bacterial cultures on agar in petri dishes. Data were expressed as zone of clearing (mm diameter +/- SEM) after overnight incubation at 35 degrees C. Significant differences (ANOVA and Turkeys test, p < 0.05) were found for each drug released from the disc of fibrin sealant compared with other vehicles. Release from filter paper discs compared with nitrocellulose was significant for nitrofurazone and norfloxacin. Serial transfer of fibrin discs to fresh bacterial cultures after 24 hours showed no zones of clearing. The data show that fibrin sealant releases topical drugs with no inhibition of antimicrobial activity on burn organisms. Greater zones of clearing from fibrin sealant may result from passive fluid retention or from active binding to fibrin followed by protease digestion by burn organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Attachment of an aminoglycoside, amikacin, to implantable collagen for local delivery in wounds.

Cultured skin substitutes consisting of implantable collagen (COL) and cultured human skin cells often fail clinically from destruction by microbial contamination. Hypothetically, addition of selected antimicrobial drugs to the implant may control microbial contamination and increase healing of skin wounds with these materials. As a model for drug delivery, bovine skin COL (1 mg/ml) and amikacin (AM; 46 micrograms/ml) were modified by covalent addition of biotin (B-COL and B-AM, respectively) from B-N-hydroxysuccinimide and bound together noncovalently with avidin (A). B-COL was incubated with A and then with B-peroxidase (B-P) or by serial incubation with B-AM and B-P, before P-dependent chromogen formation. Colorimetric data (n = 12 per condition) from spot tests on nitrocellulose paper were collected by transmission spectrophotometry. Specificity of drug binding in spot tests was determined by (i) serial dilution of B-COL; (ii) reactions with COL, AM, or P that had no B; (iii) removal of A; or (iv) preincubation of B-COL-A with B before incubation with B-P. Binding of B-AM was (i) dependent on the concentration of B-COL; (ii) specific to B-COL, A, and B-P (P < 0.05); and (iii) not eluted by incubation in 0.15 or 1.0 M NaCl. B-AM was found to block binding of B-P to the B-COL-A complex and to retain bacteriocidal activity against 10 clinical isolates of wound bacteria in the wet disc assay. Antimicrobial activity of B-AM was removed from solution by treatment with magnetic A and a permanent magnet. These results suggest that selected antimicrobial drugs can be biotinylated for attachments to COL-cultured cell implants without loss of pharmacologic activity. Because this chemistry utilizes a common ligand, any molar ratio of agents may be administered simultaneously and localized to the site of implantation. Images

Improvement of Epidermal Barrier Properties in Cultured Skin Substitutes after Grafting onto Athymic Mice

Barrier function in cultured skin substitutes (CSS) prepared from human cell sources was measured by noninvasive (surface hydration, transepidermal water loss) and invasive methods (water permeation, niacinamide flux) before and after grafting onto athymic mice. In vitro measurements were made on days 7 and 14. Although three of the four measures of barrier function improved markedly from day 7 to 14, the values obtained were still far from those obtained with native human skin controls. Additional CSS were grafted onto athymic mice on day 14, and skin was harvested 2 and 6 weeks after grafting. Grafting brought about a substantial decrease in all measurements by 2 weeks and almost complete normalization of barrier function after 6 weeks. The most sensitive measure of this recovery was niacinamide permeability, which decreased from (280 ± 40) × 10–4 cm/h in vitro to (17 ± 30) × 10–4 cm/h 2 weeks after grafting and (5 ± 2) × 10–4 cm/h 6 weeks after grafting, versus control values of (2 ± 2) × 10–4 cm/h in human cadaver skin and (0.6 ± 0.4) × 10–4 cm/h in human epidermal membrane prepared from freshly excised breast skin. These results demonstrate the reformation of epidermal barrier function after transplantation and provide insights for the development of a functional epidermal barrier in CSS in vitro.